The objective of this application is to monitor the activation and co-stimulation of genetically altered human T-lymphocytes containing chimeric antigen receptors (CARs). T-cells can be genetically engineered to ensure their therapeutic efficacy, and to express markers that can be tracked by non-invasive imaging. Genetic labeling of lymphocytes with nuclear or optical reporter genes allows for non-invasive imaging of T-cell fate in both pre-clinical cancer models and in patients with cancer. This proposal focuses on prostate cancer, based on the genetic targeting of prostate-specific membrane antigen (PSMA). The objectives we are pursuing will increase our knowledge about the dynamics of T-cell activation, co-stimulation and duration of anti-tumor response in vivo. Specifically, using reporter gene imaging in vivo we will explore the role of selected pro- survival/proliferation co-stimulatory pathways which augment the therapeutic potency of genetically targeted T-cells. We hypothesize that genetic labeling of T lymphocytes with novel pathway-specific reporter constructs will allow for repetitive non-invasive monitoring of activation and co-stimulation in T-cells and provide an early assessment of treatment outcome during PSMA-specific adoptive T-cell immunotherapy. The role of imaging will be essential in gaining a better understanding of the duration and magnitude of T-cell activation, persistence and T-cell proliferation. The combined use of therapeutic CARs and imaging reporter genes will provide an innovative research technique and a powerful novel clinical tool in cancer immunotherapy.